Succinic acid production with Actinobacillus succinogenes: rate and yield analysis of chemostat and biofilm cultures

Abstract

BackgroundSuccinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof.ResultsThe volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g-1, while the ratios for biofilm runs started at 2.4 g.g-1 and increased up to 3.3 g.g-1 as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g-1 for chemostat runs, while the yields varied between 0.63 g.g-1 and 0.74 g.g-1 for biofilm runs. Specific growth rates (μ) were shown to be severely inhibited by the formation of organic acids, with μ only 12% of μmax at a succinic acid titre of 7 g.L-1. Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases.ConclusionsThe novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes

“…Similar to previous continuous, biofilm studies on A. succinogenes Bradfield and Nicol, 2016;Brink and Nicol, 2014;Maharaj et al, 2014), in the current study, the succinic acid yield on substrate ( Figure 2A) and the ratio of succinic acid to acetic acid (Y AASA ; Figure 2B) increased with increasing succinic acid titre (C SA ). Furthermore, the formic acid-to-acetic acid ratio (Y AAFA ) decreased with increasing C SA ( Figure 2B), indicating increased pyruvate-or formate dehydrogenase activity It has been suggested that these trends are linked to a titre-dependent shift in metabolism from a growth to a non-growth or stationary mode Brink and Nicol, 2014), with associated incomplete closures of mass and redox balances.…”

“…DCWs were performed on the fermentation broth after biofilm scrubbing (see below) to determine the biomass content present in the biofilm sample and the enzyme assays. Attempts to separate cells and extracellular polymeric substance from the biofilm showed poor repeatability compared to a previous study (Brink and Nicol, 2014). Therefore, the DCW of scrubbed biofilm was used as a measure of the total biomass content of each sample and was assumed to be an accurate representation of the overall biomass content in the reactor.…”

“…Furthermore, the formic acid-to-acetic acid ratio (Y AAFA ) decreased with increasing C SA ( Figure 2B), indicating increased pyruvate-or formate dehydrogenase activity It has been suggested that these trends are linked to a titre-dependent shift in metabolism from a growth to a non-growth or stationary mode Brink and Nicol, 2014), with associated incomplete closures of mass and redox balances. In the current study, average mass balance closures ( Figure 3A) were 91% and 84% for glucose and xylose respectively and decreased with increasing C SA , which is in agreement with previous observations.…”

“…However, it has been shown that succinic acid production occurs in preference to the two byproducts at elevated succinic-and total acid concentrations under continuous biofilm conditions, resulting in an increase in the succinic acid yield on substrate Brink and Nicol, 2014). The improvement in yield has been linked to a shift in metabolic flux distribution where the organism enters a non-growth or stationary phase (Brink and Nicol, 2014).…”

“…The improvement in yield has been linked to a shift in metabolic flux distribution where the organism enters a non-growth or stationary phase (Brink and Nicol, 2014). Furthermore, mass and redox balance closures based on excreted metabolites, have been shown to be incomplete under these conditions.…”